Many biologically interesting proteins are glycosylated at their asparagine, serine, and threonine residues. Glycosylation has now been recognized as being more ubiquitous and structurally varied than all other types of post-translational modifications combined. While glycoproteins are being increasingly implicated to be crucial to processes as diverse as cellular adhesion, egg fertilization, targeting aging cells, etc., very little is known about the underlying molecular basis of sugar-sugar and sugar-protein interactions. The extreme complexity of glycan structures, multiple substitutions (microheterogeneity) at glycosylation sites, and the structural diversity associated with the protein backbone itself thus represent an enormous task for analytical structural studies.
Traditionally, a complete structural analysis of the glycan structures, including determination of the carbohydrate sequence and sugar linkage forms, has been a tedious, multitechnique task, often necessitating milligram to gram quantities of material. To progress from this situation, it is first essential to have a sensitive end-measurement methodology.
Modern mass spectrometry (MS), featuring most prominently the matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) methodologies, has now become prominent in structural elucidation of complex carbohydrates. Mechref, Y. et al., Chem. Rev. 102, 321-370 (2002); Dell, A. et al., Science 291, 2351-2356 (2001). During studies on complex carbohydrate mixtures (e.g., various glycan pools released from glycoproteins), a separation in time and space prior to MS, in either on-line or off-line modes, appears necessary. While MS of separated glycans is inherently informative of the molecular details such as sequence and linkage, certain chromatographic and electromigration principles can also readily resolve various isomeric structures.
The gradual development of high-performance liquid chromatography (HPLC) over the last three decades, with an ever-expanding range of new sorption materials, has provided some viable alternatives in carbohydrate separations. Hydrophilic interaction and adsorption chromatography, reversed-phase HPLC, anion-exchange chromatography, and lectin affinity column chromatography have all been used (Mechref, Y. et al., Chem. Rev. 102, 321-370 (2002)), albeit in different analytical applications and detection modes and, in some cases, with the use of sample derivatization.
The HPLC carbohydrate methodologies developed to date are rarely suitable for a coupling to MS: the silica-based hydrophilic interaction and adsorption columns suffer from lack of reproducibility and short lifetimes, while the additional HPLC modes requiring strong buffers and alkaline conditions are not easily adjustable to MS operation. Moreover, the extraordinary complexity of most glycan mixtures presents a major challenge to the ordinary HPLC with a limited resolving power. The modern capillary electromigration techniques, such as capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC), and capillary electrochromatography (CEC), were recently shown to provide unprecedented resolution of complex oligosaccharide mixtures. Mechref, Y. et al., Chem. Rev. 102, 321-370 (2002); Novotny, M., Methods Enzymol. 271, 319-347 (1996); Hong, M. et al., Anal. Chem. 70, 568-573 (1998). However, CZE and MEKC also suffer from certain analytical disadvantages, namely, (a) incompatibility of their separation buffers and mobile phases with MS, (b) limited options for sample preconcentration, and (c) on occasion, a lack of separation selectivity. Due to its potential for variations in the design of suitable mobile-phase/stationary phase combinations, CEC is perhaps the most suitable electromigration alternative for coupling with MS methodologies.